Prolyl hydroxylase (PHD2) and `factor inhibiting HIF' (FIH) are the primary O2-sensors in human cells. PHD2 and FIH are Fe(II)- ?KG-dependent dioxygenases, catalyzing the oxidative decarboxylation of ?KG to produce succinate and CO2 to generate a ferryl oxidant, which hydroxylates specific amino acid residues on the hypoxia inducible factor (HIF) - PHD2 and FIH turn off HIF-controlled gene expression in response to increases in [O2]. The key to sensing O2 is to ensure that O2 activation only occurs after the substrate (HIF) binds to the enzyme. Contacts involved in substrate-triggered O2-activation are poorly understood for the HIF hydroxylases, as well as for the broader class of ?KG-dependent dioxygenases, yet are key to O2-sensing by HIF hydroxylases. The central hypothesis of this proposal is that changes in the second coordination sphere are induced by substrate binding, and these changes increase the rate of ligand exchange, and the O2-affinity at the cofactor. We will test this model using steady-state kinetic probes coupled with electronic spectroscopy and crystallography to correlate reactivity with Fe(II) geometry. These mechanistic studies will increase our understanding of O2 activation by the broader class of ?KG oxygenases, as well as providing specific insight into the reactivity of the HIF hydroxylases. We will extend this understanding into engineering of FIH to perform radical rebound chemistry leading to new C-X bond forming reactions.